Technical Abstract:
Our goal is to further characterize infection pathways in barley spikes and to analyze whether and how infection patterns are affected in both widely used wild types and transgenic resistance sources. Here, we present evidence of infection pathways in cv Conlon (susceptible) using the previously reported GFP-expressing GZT501 F. graminearum strain and visualizing its green fluorescence with a stereoscope, an epifluoresence and a confocal microscope. Localized inoculations with very low concentration conidial suspensions were performed both ex planta and in planta. Detached palea, lemma and kernels placed on 0.7% water agar plates were inoculated with 2 ml drops containing 5-20 conidia on either adaxial or abaxial faces (palea, lemma), and dorsal or ventral faces (kernels). After 48 h at 21°C, the presence/absence of fungal colonies was assessed. In planta inoculation was performed by depositing 2 ml drops containing ca. 5 conidia on the tip of individual florets, within the space defined by the brush hair region of the kernel and the adaxial faces of both palea and lemma tips at early dough stages. Spikes were then covered for two days with a plastic bag. Inoculated spikelets were sampled at 6 days after inoculation (dai). All experiments included a water-inoculation treatment as a control. Fungal colonies were more frequent on adaxial inoculated than abaxial inoculated paleas (9/9 vs. 4/10) and lemmas (7/10 vs. 3/10). In all cases, colonies consisted of sparse superficial hyphae. At 2 dai ex planta, no evidence of fungal penetration and invasion was observed in cross-sectioned tissues. Conversely, fungal colonies were frequent in both ventral (12/15) and dorsal (10/15) inoculated detached kernels. Abundant intercellular hyphae were readily observed within pericarp cells of cross sectioned kernels. At 6 dai in planta, fluorescence was localized at the upper two thirds of the inoculated spikelet and closely associated with discoloration of palea, lemma and kernel. Very few superficial hyphae were observed on the abaxial faces of lemmas and paleas. Little or no discoloration or fluorescent hyphae were observed in associated glumes and adjacent empty spikelets and rachilla. However, several fluorescent hyphae emerging from the infected floret tip started colonizing the spikelet immediately above as well as adjacent green tissues, including glumes and empty spikelets. Most of the macroscopically observed fluorescence resulted from dense superficial mycelia growing in the space between the pericarp and the adaxial surface of the palea and lemma. Fungal penetration and intra and intercellular invasion was observed in both palea and lemma cross-sections from the adaxial thin cell wall epidermis and parenchyma towards the abaxial thick cell wall parenchyma. Kernel cross-sections showed massive intercellular hyphae along pericarp epidermis and parenchyma and cross-cell layers. Our preliminary results show that invasion of pericarp occurred earlier and more abundantly than lemmas and paleas, and that adaxial surfaces of palea and lemma are the candidate primary sites of invasion in these tissues. These findings support a model in which early fungal development occurs towards the inside of the kernel, then towards the kernel surface generating a dense mass of mycelia, which in close contact with adaxial surfaces of the paleas and lemmas may start second wave of invasion. A third wave may occur when running hyphae start exploring adjacent green tissues in the spikes. In accordance with these results, we will target the monitoring of early invasion at the upper pericarp tissue and at the adaxial faces of the lemmas and paleas.